CE7-specific chimeric T cell receptor

ABSTRACT

Genetically engineered, CE7-specific redirected immune cells expressing a cell surface protein having an extracellular domain comprising a receptor which is specific for CE7, an intracellular signaling domain, and a transmembrane domain, and methods of use for such cells for cellular immunotherapy of CE7+ neuroblastoma are disclosed. In one embodiment, the immune cell is a T cell and the cell surface protein is a single chain FvFc: ζ receptor where Fv designates the V H  and V L  chains of a single chain monoclonal antibody to CE7 linked by peptide, F c  represents a hinge-C H 2-C H 3 region of a human IgG 1 , and ζ represents the intracellular signaling domain of the zeta chain of human CD3. DNA constructs encoding a chimeric T-cell receptor and a method of making a redirected T cell expressing a chimeric T cell receptor by electroporation using naked DNA encoding the receptor are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/120,198 filed 11 Apr. 2002, now U.S. Pat. No. 7,070,995, Ser. No.10/120,198 is related to claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application Ser. No. 60/282,859 filed 11 Apr. 2001.Each application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to the field of genetically engineered,redirected immune cells and to the field of cellular immunotherapy ofCE7 malignancies.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice are incorporated by reference.

Neuroblastoma, a neoplasm arising from sympathetic ganglion cells, isthe most common extracranial solid tumor of childhood and is third inincidence among pediatric malignancies after the leukemia-lymphomasyndromes and central nervous system tumors [1-2]. Approximately 550 newcases occur annually in the United States; seventy-nine percent ofchildren are diagnosed prior to their fifth birthday. Stage of diseaseat diagnosis, patient's age at diagnosis, and characteristics of tumorcells such as histologic appearance and NMYC gene amplification areimportant prognostic factors which can be utilized to categorizepatients into low, intermediate and high risk for poor outcome [3, 4].While survival for low and intermediate risk neuroblastoma is excellent,the prognosis for high-risk neuroblastoma remains dismal. Despiteimproved tumor response rates following intensive multi-modality therapythe median response duration of high risk tumors is less than 1 year andless than 40% of patients with high risk neuroblastoma survive more than2 years [3-6]. To date there are no treatment modalities with provenefficacy for salvaging children with recurrent/refractory disseminatedneuroblastoma.

Current treatment strategies for neuroblastoma are tailored torisk-stratified algorithms based on the International NeuroblastomaStaging System (INSS) [6-9]. The primary risk factors accounted for areage at diagnosis, stage of disease, tumor histology, NMYC copy number,and DNA index. High-risk disease includes those children greater thantwelve months of age with tumor dissemination (Stage IV) as well asStage 3 and 4 disease with unfavorable histology or NMYC amplification,regardless of age [10-17]. High-risk disease accounts for more than halfof newly diagnosed cases of neuroblastoma and approximatelythree-quarters of cases diagnosed in children greater than 12 months ofage. A multi-modality maximally intensive approach to treatment ofhigh-risk neuroblastoma has evolved that includes aggressive inductionchemotherapy, surgery, radiation therapy, autologous stem celltransplantation, and post-transplant biologic therapy with cis-retinoicacid. Two successive Children's Cancer Group trials (CCG-321, CCG-3891)have demonstrated improved survival for those patients receivingmyelo-ablative therapy compared to those patients receiving conventionalchemotherapy [18]. Aggressive local therapy including complete surgicalresection of the primary tumor and local radiation appears to decreasethe incidence of primary site recurrence. Unfortunately, more than 50%of all patients and 75% of patients failing to achieve a completeremission with 1^(st) line conventional therapy continue to developrecurrent disease. Relapses typically occur in a disseminated fashionwithin the first 24-months after completing front-line therapy.Responses to salvage chemotherapy are limited and generally are notdurable with only 8% of patients surviving greater that 3 years fromtime of recurrence [19-23].

Disease relapse for many children with neuroblastoma frequently occursfollowing the induction of a clinical complete response with standardtreatment modalities, demonstrating that the persistence of minimalresidual disease is a major obstacle for curative therapy. However,patients heavily treated with chemotherapy, radiation, surgery, andautologous transplantation have a limited capacity to tolerateadditional cytotoxic therapeutic modalities to target minimal residualdisease. The potential of targeting a limited tumor burden withimmune-based approaches is attractive both, because of the opportunityto invoke immunologic effector mechanisms to whichchemotherapy/radiation-resistant tumor cells are susceptible, as well asthe limited toxicity theoretically possible with tumor-specificimmunologic effector mechanisms.

Passive immunotherapy for neuroblastoma utilizing murine monoclonal andmurine/human chimeric monoclonal antibodies have focused primarily ontargeting the G_(D2) disialoganglioside present at high density on humanNB [24-26]. Cheung et al. have investigated in clinical trials theG_(D2)-specific monoclonal antibody 3F8 and have reported on the safetyand, more recently, the anti-tumor activity of antibody therapy in thesetting of minimal residual disease [27]. The long-term outcome ofpatients treated with 3F8 awaits delineation, however, limitations inits use have been observed early after treatment due to the developmentof neutralizing HAMA responses in approximately a third of patients[27]. Additionally, failure of antibody therapy to target MRD in the CNSdue to poor penetration of immunoglobulin across the blood-brain-barrierwas manifested by an unusually high incidence of isolated CNS relapsesin antibody treated patients.

The cloning and production of recombinant cytokines have facilitatedtheir introduction into clinical trials designed to activate and expandimmunologic effector cells in vivo. Various cytokines either alone or incombination have been evaluated in preclinical neuroblastoma animalmodels [28-30]. Interleukin-2 administration following transplantationhas been most extensively studied as a strategy to activate NK cells andinduce LAK cells. IL-2 therapy for patients with recurrent metastaticneuroblastoma failed to provide anti-tumor activity in 15 childrentreated [31,32]. These studies to date have revealed a significantincidence of severe toxicities associated with high-dose IL-2administration without a clear impact on decreasing disease relapse. Theprolonged use of low-dose IL-2, although not having demonstrableanti-neuroblastoma activity, can be administered to heavily pre-treatedchildren without severe toxicity [33]. Pession et al. have reported onthe administration of 212 courses of low dose IL-2 to 17 children withneuroblastoma following stem cell transplantation [33]. Thesemaintenance courses were delivered bimonthly over five days/course atIL-2 doses of 2×10⁶ U/m²/day escalating to 4×10⁶ U/m²/day. Nolife-threatening toxicities were encountered. Fever controlled byacetaminophen and transient rash were the most common side effects oftherapy. Consequently, current Phase I studies are evaluating thecombination of cytokines that activate effector cells operative inantibody dependent cellular cytotoxicity (IL-2 and GM-CSF) incombination with anti-G_(D2) antibody therapy [34,35]. Frost et al. haveinvestigated the use of monoclonal anti-GD2 antibody, 14.G2a plus IL-2in 31 children with refractory neuroblastoma. Dose limiting toxicitiesincluded generalized pain and fever without documented infection. Tumorprogression was noted in 63% of patients. Of note, 30% of patients withevaluable bone marrow disease had a significant decrease in quantity oftumor cells detected by immunohistochemical analysis [35]. Theengineering of antibody-cytokine fusion molecules appears to potentiatethe anti-tumor activity of either molecule administered separately inanimal models, these fusion proteins are currently under investigationin clinical trials [36,37].

Induction or augmentation of a cellular immune response againstneuroblastoma is an attractive strategy for eliminating resistant tumorcells. The availability of recombinant interleukin-2 (IL-2) and thedemonstration that lymphocytes cultured in high concentrations oflymphokines acquire the ability to lyse, in a non-MHC-restrictedfashion, a variety of tumor types, led to trials attempting to targetneuroblastoma with the adoptive transfer of autologous ex-vivo expandedLAK cells [38]. Up to 10¹¹ LAK cells have been administered in a singleintravenous infusion to cancer patients without dose-limiting sideeffects, demonstrating the safety of adoptive therapy with large numbersof in vitro activated autologous lymphocytes. The toxicity that has beenobserved in these trials was attributed solely to the systemic effectsof high-dose IL-2 that is required to support LAK cells in vivo [39].LAK cell therapy in children with neuroblastoma has met with significanttoxicities without obvious clinical benefit [31].

Animal models as well as a small but growing number of human tumorsystems have demonstrated that anti-tumor cellular immune responses canbe invoked or amplified by vaccination with tumor cells geneticallymodified to have enhanced immunogenicity. Transgenes that are beingevaluated for neuroblastoma tumor cell vaccines include allogeneic HLAclass II molecules, the co-stimulatory ligand B7-1, and pro-inflammatorycytokines [40-46]. Recently Bowman et al. published their results of apilot study in which ten children with relapsed advanced stageneuroblastoma were treated with autologous tumor cells geneticallymodified to secrete IL-2 [47]. Of note, five patients had objectivesystemic anti-tumor responses correlating with the development of invitro detected anti-tumor cellular cytotoxicity. These studies provide aglimpse at the potential of cellular immunotherapy for neuroblastoma butunderscore the variability of inducing clinically relevant anti-tumorresponses with vaccines and the technical difficulties in generatingautologous genetically manipulated tumor cell lines for thisapplication.

Antigen-specific T cells are immunologic effector cells that conferprotection from lethal tumor challenge in animal models [48]. Adoptivetransfer of tumor-specific T cell clones into tumor bearing hosts caneradicate established disseminated tumors. Enomoto et al. havedemonstrated in a murine model system employing a poorly immunogenicsyngeneic neuroblastoma, the capacity of adoptively transferredtumor-reactive cytotoxic T lymphocytes (CTL) to eradicate disseminatedneuroblastoma [49]. This provocative model system, in light of theresponses seen clinically to IL-2 producing tumor vaccineadministration, suggest that adoptive therapy withneuroblastoma-specific T cells may have significant clinical utilityprovided these T cells can be reliably isolated from this patientpopulation.

An ideal cell-surface epitope for targeting with antigen-specific Tcells would be expressed solely on tumor cells in a homogeneous fashionand on all tumors within a population of patients with the samediagnosis. Modulation and/or shedding of the target molecule from thetumor cell membrane may also impact on the utility of a particulartarget epitope for re-directed T cell recognition. To date few “ideal”tumor-specific epitopes have been defined and secondary epitopes havebeen targeted based on either lack of expression on critical normaltissues or relative over-expression on tumors. Anti-G_(D2) antibodieshave been most extensively utilized in antigen-specific immunotherapyfor neuroblastoma. G_(D2), however, is expressed on peripheral nerves aswell as brain grey matter. T cells, unlike antibody, can extensivelyaccess the CNS blood-brain barrier making G_(D2) re-directed adoptive Tcell therapy subject to potentially severe neurologic toxicities [50].

Several groups have generated murine monoclonal antibodies reactive withhuman neuroblastoma by immunization of mice with human NB tumor celllines. Blaser et al. have published on the generation of the CE7monoclonal antibody (γI/κ) raised by immunizing mice with the IMR-32human neuroblastoma cell line. CE7 uniformly binds to humanneuroblastoma cell lines and primary tumors [51-53]. This high affinityIgGI monoclonal antibody (K_(a)=10⁻¹¹) precipitates a 190-kDA plasmamembrane-associated glycoprotein [54]. Tumor cells express in excess of40,000 binding epitopes for CE7 and the target molecule does not shedfrom the cell surface. Biodistribution studies in nude mice revealedthat up to 32% of injected dose/g tissue of iodinated antibodyaccumulates in tumor explants with low blood and organ uptake [55,56].Importantly, this antibody in immunohistochemistry screening of normaltissues failed to bind to all non-neuroectodermal tissues as well asbrain [51]. Very weak binding was observed on adrenal medulla andsympathetic ganglia [50]. Carrel et al. have generated a mouse/humanchimeric antibody and are pursuing preclinical studies for developmentof CE7-targeted radioimmunotherapy for neuroblastoma [56]. As with manymonoclonal antibodies raised against tumor cell lines, the molecularidentity of the target epitope of CE7 awaits delineation.

The safety of adoptively transferring antigen-specific CTL clones inhumans was originally examined in bone marrow transplant patients whoreceived donor-derived CMV-specific T cells [57,80]. Studies from thelaboratories of Drs. Greenberg and Riddell at the Fred Hutchinson CancerResearch Center (FHCRC) have demonstrated that the reconstitution ofendogenous CMV-specific T cell responses following allogeneic bonemarrow transplantation (BMT) correlates with protection from thedevelopment of severe CMV disease [58]. In an effort to reconstitutedeficient CMV immunity following BMT, CD8⁺ CMV-specific CTL clones weregenerated from CMV seropositive HLA-matched sibling donors, expanded,and infused into sibling BMT recipients at risk for developing CMVdisease. Fourteen patients were treated with four weekly escalatingdoses of these CMV-specific CTL clones to a maximum cell dose of 10⁹cells/m² without any attendant toxicity [59]. Peripheral blood samplesobtained from recipients of adoptively transferred T cell clones wereevaluated for in vivo persistence of transferred cells. The recoverableCMV-specific CTL activity increased after each successive infusion ofCTL clones, and persisted at least 12 weeks after the last infusion.However, long term persistence of CD8⁺ clones without a concurrent CD4⁺helper response was not observed. No patients developed CMV viremia ordisease. These results demonstrate that ex-vivo expanded CMV-specificCTL clones can be safely transferred to BMT recipients and can persistin vivo as functional effector cells that may provide protection fromthe development of CMV disease.

A complication of bone marrow transplantation, particularly when marrowis depleted of T cells, is the development of EBV-associatedlymphoproliferative disease [60]. This rapidly progressive proliferationof EBV-transformed B-cells mimics immunoblastic lymphoma and is aconsequence of deficient EBV-specific T cell immunity in individualsharboring latent virus or immunologically naïve individuals receiving avirus inoculum with their marrow graft. Clinical trials conducted at St.Jude's Hospital by Rooney et al. have demonstrated that adoptivelytransferred ex-vivo expanded donor-derived EBV-specific T cell lines canprotect patients at high risk for development of this complication aswell as mediate the eradication of clinically evident EBV-transformed Bcells [61]. No significant toxicities were observed in the forty-onechildren treated with cell doses in the range of 4×10⁷ to 1.2×10⁸cells/m².

Genetic modification of T cells used in clinical trials has beenutilized to mark cells for in vivo tracking and to endow T cells withnovel functional properties. Retroviral vectors have been used mostextensively for this purpose due to their relatively high transductionefficiency and low in vitro toxicity to T cells [62]. These vectors,however, are time consuming and expensive to prepare as clinical gradematerial and must be meticulously screened for the absence ofreplication competent viral mutants [63]. Rooney et al. transducedEBV-reactive T cell lines with the NeoR gene to facilitate assessment ofcell persistence in vivo by PCR specific for this marker gene [64].Riddell et al. have conducted a Phase I trial to augment HIV-specificimmunity in HIV seropositive individuals by adoptive transfer usingHIV-specific CD8⁺ CTL clones [65]. These clones were transduced with theretroviral vector tgLS⁺HyTK which directs the synthesis of abifunctional fusion protein incorporating hygromycin phosphotransferaseand herpes virus thymidine kinase (HSV-TK) permitting in vitro selectionwith hygromycin and potential in vivo ablation of transferred cells withgancyclovir. Six HIV infected patients were treated with a series offour escalating cell dose infusions without toxicities, with a maximumcell dose of 5×10⁹ cells/m² [65].

As an alternate to viral gene therapy vectors, Nabel et al. used plasmidDNA encoding an expression cassette for an anti-HIV gene in a Phase Iclinical trial. Plasmid DNA was introduced into T cells by particlebombardment with a gene gun [66]. Genetically modified T cells wereexpanded and infused back into HIV-infected study subjects. Althoughthis study demonstrated the feasibility of using a non-viral geneticmodification strategy for primary human T cells, one limitation of thisapproach is the episomal propagation of the plasmid vector in T cells.Unlike chromosomally integrated transferred DNA, episomal propagation ofplasmid DNA carries the risk of loss of transferred genetic materialwith cell replication and of repetitive random chromosomal integrationevents.

Chimeric antigen receptors engineered to consist of an extracellularsingle chain antibody (scFvFc) fused to the intracellular signalingdomain of the T cell antigen receptor complex zeta chain (ζ) have theability, when expressed in T cells, to redirect antigen recognitionbased on the monoclonal antibody's specificity [67]. The design ofscFvFc: ζ receptors with target specificities for tumor cell-surfaceepitopes is a conceptually attractive strategy to generate antitumorimmune effector cells for adoptive therapy as it does not rely onpre-existing anti-tumor immunity. These receptors are “universal” inthat they bind antigen in a MHC independent fashion, thus, one receptorconstruct can be used to treat a population of patients with antigenpositive tumors. Several constructs for targeting human tumors have beendescribed in the literature including receptors with specificities forHer2/Neu, CEA, ERRB-2, CD44v6, and epitopes selectively expressed onrenal cell carcinoma [68-72]. These epitopes all share the commoncharacteristic of being cell-surface moieties accessible to scFv bindingby the chimeric T cell receptor. In vitro studies have demonstrated thatboth CD4⁺ and CD8⁺ T cell effector functions can be triggered via thesereceptors. Moreover, animal models have demonstrated the capacity ofadoptively transferred scFvFc: ζ expressing T cells to eradicateestablished tumors [73]. The function of primary human T cellsexpressing tumor-specific scFvFc: ζ receptors have been evaluated invitro; these cells specifically lyse tumor targets and secrete an arrayof pro-inflammatory cytokines including IL-2, TNF, IFN-g, and GM-CSF[74]. Phase I pilot adoptive therapy studies are underway utilizingautologous scFvFc: ζ-expressing T cells specific for HIV gp120 in HIVinfected individuals and autologous scFvFc: ζ-expressing T cells withspecificity for TAG-72 expressed on a variety of adenocarcinomasincluding breast and colorectal adenocarcinoma.

Investigators at City of Hope have engineered a CD20-specific scFvFc: ζreceptor construct for the purpose of targeting CD20+ B-cell malignancy[75]. Preclinical laboratory studies have demonstrated the feasibilityof isolating and expanding from healthy individuals and lymphomapatients CD8+ CTL clones that contain a single copy of unrearrangedchromosomally integrated vector DNA and express the CD20-specificscFvFc: ζ receptor [76]. To accomplish this, purified linear plasmid DNAcontaining the chimeric receptor sequence under the transcriptionalcontrol of the CMV immediate/early promoter and the NeoR gene under thetranscriptional control of the SV40 early promoter was introduced intoactivated human peripheral blood mononuclear cells by exposure of cellsand DNA to a brief electrical current, a procedure calledelectroporation [77]. Utilizing selection, cloning, and expansionmethods currently employed in FDA-approved clinical trials, genemodified CD8+ CTL clones with CD20-specific cytolytic activity have beengenerated from each of six healthy volunteers in 15 separateelectroporation procedures [76]. These clones when co-cultured with apanel of human CD20+ lymphoma cell lines proliferate, specifically lysetarget cells, and are stimulated to produce cytokines.

It is desired to develop additional redirected immune cells and, in apreferred embodiment redirected T cells for treating neuroblastoma andother malignancies expressing the CE7 recognized target epitope.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides genetically engineeredimmune cells (referred to herein as CE7-specific redirected immunecells) which express and bear on the cell surface membrane aCE7-specific chimeric immune receptor (referred to also as CE7R)comprising an intracellular signaling domain, a transmembrane domain(TM) and a CE7-specific extracellular domain (domain derived from thevariable heavy and light chain regions of the CE7 monoclonal antibody).The present invention also provides the CE7-specific chimeric immunereceptors, DNA constructs encoding the receptors, and plasmid expressionvectors containing the constructs in proper orientation for expression.

In a second aspect, the present invention provides a method of treatinga CE7⁺ malignancy in a mammal (i.e., those malignancies which expressthe CE7 recognized target epitope) which comprises administeringCE7-specific redirected immune cells to the mammal in a therapeuticallyeffective amount. In one embodiment, CD8⁺ CE7-specific redirected Tcells are administered, preferably with CD4⁺ CE7-specific redirected Tcells. In a second embodiment, CD⁺4CE7-specific redirected T cells areadministered to a mammal (preferably in combination with CD8⁺ cytotoxiclymphocytes which express the CE7-specific chimeric receptor).

In a third aspect, the present invention provides a method of making andexpanding the CE7-specific redirected T cells which comprisestransfecting T cells with an expression vector containing a DNAconstruct encoding the CE7-specific chimeric receptor, then stimulatingthe cells with CE7+cells, recombinant CE7, or an antibody to thereceptor to cause the cells to proliferate. In one embodiment, theredirected T cells are prepared by electroporation. In a secondembodiment, the redirected T cells are prepared by using viral vectors.

In another aspect, the invention provides genetically engineered stemcells which express on their surface membrane a CE7-specific chimericimmune receptor having an intracellular signaling domain, atransmembrane domain and a CE7-specific extracellular domain.

In another aspect, the invention provides genetically engineered naturalkiller(NK) cells which express on their surface membrane a CE7-specificchimeric immune receptor having an intracellular signaling domain, atransmembrane domain and a CE7-specific extracellular domain.

In another aspect, the invention provides genetically engineeredneutrophils which express on their surface membrane a CE7-specificchimeric immune receptor having an intracellular signaling domain, atransmembrane domain and a CE7-specific extracellular domain.

In yet another aspect, the invention provides genetically engineeredmacrophage which express on their surface membrane a CE7-specificchimeric immune receptor having an intracellular signaling domain, atransmembrane domain and a CE7-specific extracellular domain.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G and 1H show the double-stranded DNAsequence of the plasmid containing a CE7R chimeric immunoreceptor of thepresent invention and show the source of the DNA found in the plasmid.The amino acid sequences of the CE7R/HyTK are also shown. The sensestrand of the double-stranded DNA sequence is set forth in SEQ ID NO:5.The sense strand of the double stranded sequence coding for the CE7Rchimeric receptor is set forth in SEQ ID NO:1, and the correspondingamino acid sequence is set forth in SEQ ID NO:2. The sense strand of thedouble stranded sequence coding for HyTK is set forth in SEQ ID NO:3,and the corresponding amino acid sequence is set forth in SEQ ID NO:4.

FIG. 2A is a schematic representation of a CE7R/scFvFc: ζ chimericreceptor.

FIG. 2B is a schematic representation of the plasmid pMG-CE7R/HyTK; thesequence which is shown in FIG. 1 A-H.

FIG. 3 shows Western blot analyses which demonstrate the expression ofthe CE7R/scFvFc: ζ chimeric receptor.

FIGS. 4A and 4B show the results of Fluorescent Activated Cell Sortingwhich demonstrate the cell surface location of the CE7R/scFvFc: ζchimeric receptor. Flow cytometric analysis of transfected T-cellsreacted with anti-murine FAB which react with the CE7 portion of theCE7R and anti-human Fc specific antibodies which react with the IgGportion of the CE7R, confirmed the cell-surface expression of the CE7RscFvFc: ζ on T cell transfectants (FIG. 4B) as evidenced bycosegregation of the CE7R antigens with known T cell antigens (FIG. 4A).

FIG. 5 is a graphical representation which shows the production of IL-2by Jurkat T-cells expressing the CE7R/scFvFc: ζ chimeric receptor thatare co-cultured with neuroblastoma cells.

FIG. 6 A-D are graphical representations showing the anti-neuroblastomaactivity of primary human CD8⁺ cytotoxic T lymphocytes expressing theCE7R/scFvFc: ζ chimeric receptor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to genetically engineered, redirectedimmune cells and to their use for cellular immunotherapy of malignancieswhich express the CE7 recognized target epitope, including, but notlimited to, neuroblastoma.

In one aspect, the present invention provides genetically engineered Tcells which express and bear on the cell surface membrane a CE7-specificchimeric T-cell receptor having an intracellular signaling domain, atransmembrane domain and a CE7-specific extracellular domain. Theextracellular domain comprises a CE7-specific receptor. Individual Tcells of the invention may be CD4⁺/CD8⁻, CD4⁻/CD8⁺, CD4⁻/CD8⁻ orCD4⁺/CD8⁺. The T cells may be a mixed population of CD4⁺/CD8⁻ andCD4⁻/CD8⁺ cells or a population of a single clone. CD4⁺ T cells of theinvention produce IL-2 when co-cultured in vitro with CE7⁺ neuroblastomacells. CD8⁺ T cells of the invention lyse CE7⁺ human neuroblastomatarget cells when co-cultured in vitro with the target cells. Theinvention further provides the CE7-specific chimeric T-cell receptors,DNA constructs encoding the receptors, and plasmid expression vectorscontaining the constructs in proper orientation for expression.

In a preferred embodiment, the CE7-specific redirected T cells expressCE7-specific chimeric receptor scFvFc: ζ, where scFv designates theV_(H) and V_(L) chains of a single chain monoclonal antibody to CE7, Fcrepresents at least part of a constant region of an IgG₁, and ζrepresents the intracellular signaling domain of the zeta chain of thehuman CD3 complex. The extracellular domain scFvFc and the intracellulardomain ζ are linked by a transmembrane (TM) domain such as thetransmembrane domain of CD4. In a specific preferred embodiment, a fulllength scFvFc: ζ cDNA, designated CE7R, comprises the human GM-CSFreceptor alpha chain leader peptide, CE7 V_(H), Gly-Ser linker, CE7V_(L), human IgG₁ Fc, human CD4 TM, and human cytoplasmic zeta chain.“Chimeric TCR” means a receptor which is expressed by T cells and whichcomprises intracellular signaling, transmembrane and extracellulardomains, where the extracellular domain is capable of specificallybinding in an HLA unrestricted manner an antigen which is not normallybound by a T cell receptor in that manner. Stimulation of the T cells bythe antigen under proper conditions results in proliferation (expansion)of the cells and/or production of cytokines (e.g., IL-2) and/orcytolysis.

In a second aspect, the present invention provides a method of treatinga CE7⁺ malignancy in a mammal which comprises administering CE7-specificredirected T cells to the mammal in a therapeutically effective amount.In one embodiment of this aspect of the invention, a therapeuticallyeffective amount of CD8⁺ CE7-specific redirected T cells areadministered to the mammal. The CD8⁺ T cells are preferably administeredwith CD4⁺ CE7-specific redirected T cells. In a second embodiment ofthis aspect of the invention, a therapeutically effective amount of CD4⁺CE7-specific redirected T cells are administered to the mammal. The CD4⁺T cells are preferably administered with CD8⁺ T CE7-specific redirectedT cells.

In a third aspect, the present invention provides a method of making andexpanding the CE7-specific redirected T cells which comprisestransfecting T cells with an expression vector containing a DNAconstruct encoding the CE7-specific chimeric receptor, then stimulatingthe cells with CE7⁺ cells, recombinant CE7, or an antibody to thereceptor to cause the cells to proliferate. According to this aspect ofthe present invention, the method preferably stably transfects andre-directs T cells using electroporation of naked DNA. Alternatively,viral vectors carrying the heterologous genes are used to introduce thegenes into T cells. By using naked DNA, the time required to produceredirected T cells can be significantly reduced. “Naked DNA” means DNAencoding a chimeric T cell receptor (TCR) contained in an expressioncassette which comprises the structural gene for the chimeric T cellreceptor to which is attached regulatory DNA regions (promoter,enhancer, polyadenylkation site and the like) that permit expression ofthe gene in transfected cells. The naked DNA may further be covalentlybound to plasmid DNA as a DNA delivery or expression vector. Theelectroporation method of this invention produces stable transfectantswhich express and carry on their surfaces the chimeric TCR (cTCR).

In a preferred embodiment of the transfection method of the invention,the T cells are primary human T cells, such as human peripheral bloodmononuclear cells (PBMC), which have previously been consideredresistant to stable transfection by electroporation of plasmid vectors.Preferred conditions include the use of DNA depleted of endotoxin andelectroporation within about 3 days following mitogenic stimulation of Tcells. Following transfection, the transfectants are cloned and a clonedemonstrating presence of a single integrated unrearranged plasmid andexpression of the chimeric receptor is expanded ex vivo. The cloneselected for expansion preferably is CD8⁺ and demonstrates the capacityto specifically recognize and lyse neuroblastoma target cells whichexpress the target epitope of CE7. The clone is expanded by stimulationwith IL-2 and preferably another stimulant which is specific for thecTCR.

The invention is described herein primarily with reference to thespecific scFvFc: ζ construct and receptor of SEQ ID NOs:1 and 2, but theinvention is not limited to that specific construct and receptor. ThescFv portion can be replaced by any number of different CE7 bindingdomains, ranging from a minimal peptide binding domain, to a structuredCE7 binding domain from a phage library, to antibody like domains usingdifferent methods to hold the heavy and light chain (or peptide-bindingdomains of each) together. The arrangement could be multimeric such as adiabody. It is possible that the T cell receptor variant is also amultimer. Multimers are most likely caused by cross pairing of thevariable portion of the light and heavy chains into what has beenreferred to by Winters as a diabody.

The hinge portion of the construct can have multiple alternatives frombeing totally deleted, to having the first cysteine maintained, to aproline rather than a serine substitution, to being truncated up to thefirst cysteine. The Fc portion of IgG₁ can be deleted or replaced withthe Fc portion of IgG₄, although there is data to suggest that thereceptor preferably extends from the membrane. Any protein which isstable and dimerizes can serve this purpose. One could use just one ofthe Fc domains, e.g, either the C_(H)2 or C_(H)3 domain.

Alternatives to the CD4 transmembrane domain include the transmembraneCD3 zeta domain, or a cysteine mutated CD3 zeta domain, or othertransmembrane domains from other transmembrane signaling proteins suchas CD16 and CD8. The CD3 zeta intracellular domain was taken foractivation. Intracellular signaling portions of other members of thefamilies of activating proteins can be used, such as FcγRIII and FcεRI.See Gross et al. [78], Stancovski et al. [68], Moritz et al. [70], Hwuet al. [79], Weijtens et al. [74], and Hekele et al. [71], fordisclosures of cTCR's using these alternative transmembrane andintracellular domains.

Additional cytoplasmic domains which are known to augment lytic activityare contemplated by the present invention. Such additional cytoplasmicdomains may be selected from the group including CD28 and 4-1BB. Thecytoplasmic domains may be used together on a chimeric immune receptorarranged in “molecular series” or alternatively as distinct scFvFcconstructs co-expressed on a redirected immune cell.

Cellular Immunotherapy Using Redirected T Cells

The strategy of isolating and expanding antigen-specific T cells as atherapeutic intervention for human disease has been validated inclinical trials [59, 80, 81]. Initial studies have evaluated the utilityof adoptive T cell therapy with CD8⁺ cytolytic T cell (CTL) clonesspecific for cytomegalovirus-encoded antigens as a means ofreconstituting deficient viral immunity in the setting of allogeneicbone marrow transplantation and have defined the principles andmethodologies for T cell isolation, cloning, expansion and re-infusion[80]. A similar approach has been taken for controlling post-transplantEBV-associated lymphoproliferative disease. EBV-specific donor-derived Tcells have the capacity to protect patients at high risk for thiscomplication as well as eradicate clinically evident disease whichmimics immunoblastic B cell lymphoma [81]. These studies clearlydemonstrate that adoptively transferred ex vivo expanded T cells canmediate antigen-specific effector functions with minimal toxicities andhave been facilitated by targeting defined virally-encoded antigens towhich T cell donors have established immunity.

The application of adoptive T cell therapy as a treatment modality forhuman malignancy has been limited by the paucity of molecularly-definedtumor antigens capable of eliciting a T cell response and the difficultyof isolating these T cells from the tumor-bearing host. Consequently,initial cellular immunotherapy trials utilizing autologous antitumoreffector cells relied on antigen nonspecific effector cells such aslymphokine activated killer (LAK) cells which had limited efficacy andpronounced toxicities [82, 83]. In an attempt to enhance thetumor-specificity of infused effector cells, IL-2 expandedtumor-infiltrating lymphocytes (TIL) were evaluated [84]. Responses toTIL infusions were sporadic due in part to the heterogeneous populationof cells expanded with unpredictable antitumor specificities. Patientswith melanoma and renal cell carcinoma however occasionally manifestedstriking tumor regressions following TIL infusions and tumor-specificMHC-restricted T cell clones have been isolated from these patients.Recently, expression cloning technologies have been developed toidentify the genes encoding tumor antigens thereby facilitating thedevelopment of recombinant DNA-based vaccine strategies to initiate oraugment host antitumor immunity, as well as in vitro culture systems forgenerating tumor-specific T cells from cancer patients [85]. Clinicaltrials utilizing autologous tyrosinase-specific CTL for the treatment ofmelanoma are currently underway and will likely provide major insightsinto the efficacy of targeting tumors with antigen-specificMHC-restricted T cell clones.

Endowing T cells with a desired antigen specificity based on geneticmodification with engineered receptor constructs is an attractivestrategy since it bypasses the requirement for retrievingantigen-specific T cells from cancer patients and, depending on the typeof antigen recognition moiety, allows for targeting tumor cell-surfaceepitopes not available to endogenous T cell receptors. Studies to definethe signaling function of individual components of the TCR-CD3 complexrevealed that chimeric molecules with intracellular domains of the CD3complex's zeta chain coupled to extracellular domains which could becrosslinked by antibodies were capable of triggering biochemical as wellas functional activation events in T cell hybridomas [86]. Recentadvances in protein engineering have provided methodologies to assemblesingle chain molecules consisting of antibody variable regions connectedby a flexible peptide linker which recapitulate the specificity of theparental antibody [87, 88]. Several groups have now reported on thecapacity of chimeric single chain receptors consisting of anextracellular scFv and intracellular zeta domain to re-direct T cellspecificity to tumor cells expressing the antibody's target epitope;receptor specificities have included HER2/Neu, and less wellcharacterized epitopes on renal cell and ovarian carcinoma [68, 70, 71,74, 78, 79]. An idiotype-specific scFv chimeric TCR has been describedwhich recognizes the idiotype-expressing lymphoma cell's surfaceimmunoglobulin as its ligand [78]. Although this approach swaps a lowaffinity MHC-restricted TRC complex for a high affinity MHC-unrestrictedmolecular linked to an isolated member of the CD3 complex, thesereceptors do activate T cell effector functions in primary human T cellswithout apparent induction of subsequent anergy or apoptosis [74].Murine model systems utilizing scFvcζ transfected CTL demonstrate thattumor elimination only occurs in vivo if both cells and IL-2 areadministered, suggesting that in addition to activation of effectorfunction, signaling through the chimeric receptor is sufficient for Tcell recycling [71].

Although chimeric receptor re-directed T cell effector function has beendocumented in the literature for over a decade, the clinical applicationof this technology for cancer therapy is only now beginning to beapplied. ex vivo expansion of genetically modified T cells to numberssufficient for re-infusion is required for conducting clinical trials.Not only have sufficient cell numbers been difficult to achieve, theretention of effector function following ex vivo expansion has not beenroutinely documented in the literature.

Treatment of CE7⁺ Malignancies with CE7-Specific Redirected T Cells

This invention represents the targeting of a universal neuroblastomacell-surface epitope with CE7-specific redirected T cells. Neuroblastomacells are an excellent target for redirected T cells, as they expressthe CE7 target epitope antigen. CE7 target epitope is an ideal targetepitope for recognition by CE7-specific redirected T cells due to theprevalence of CE7⁺ disease, the uniformity of expression by tumor cells,and the stability of the CE7 target epitope molecule on the cellsurface.

We have found that expansion of CE7 specific re-directed CD8⁺ CTL cloneswith OKT3 and IL-2 routinely results in the generation of greater than10⁹ cells over a period of approximately six weeks, and that the clonesretain their effector function following expansion, as shown byfunctional chromium release assay data. Our observation that theplasmid/scFvFc: ζ system can generate transfectants with disruptedplasmid sequence underscores the desirability of cloning transfectantsand expanding those clones demonstrating the presence of a singleunrearranged integrated plasmid, expression of the chimeric receptor,and the capacity to specifically recognize and lyse CE7⁺ neuroblastomatarget cells.

Equipping T cells with a suicide gene such as the herpes virus thymidinekinase gene allows for in vivo ablation of transferred cells followingadoptive transfer with pharmacologic doses of gancyclovir and is astrategy for limiting the duration or in vivo persistence of transferredcells [89].

Patients can be treated by infusing therapeutically effective doses ofCD8⁺ CE7-specific redirected T cells in the range of about 10⁶ to 10¹²or more cells per square meter of body surface (cells/m²). The infusionwill be repeated as often and as many times as the patient can tolerateuntil the desired response is achieved. The appropriate infusion doseand schedule will vary from patient to patient, but can be determined bythe treating physician for a particular patient. Typically, initialdoses of approximately 10⁹ cells/m² will be infused, escalating to 10¹⁰or more cells/m². IL-2 can be co-administered to expand infused cellspost-infusion. The amount of IL-2 can about 10³ to 10⁶ units perkilogram body weight. Alternatively or additionally, an scFvFc:ζ-expressing CD4⁺ T_(H1) clone can be co-transferred to optimize thesurvival and in vivo expansion of transferred scFvFc: ζ-expressing CD8⁺T cells.

The dosing schedule may be based on Dr. Rosenberg's published work[72-73] or an alternate continuous infusion strategy may be employed.CE7-specific redirected T cells can be administered as a strategy tosupport CD8⁺ cells.

It is known that chimeric immune receptors are capable of activatingtarget-specific lysis by phagocytes, such as neutrophils and NK cells,for example (90). Thus, the present invention also contemplates the useof chimeric T-cell receptor DNA to transfect into non-specific immunecells including neutrophils, macrophages and NK cells. Furthermore, thepresent invention contemplates the use of chimeric T-cell receptor DNAto transfect stem cells prior to stem cell transplantation procedures.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.[See, e.g., 91-108].

EXAMPLES

The present invention is further detailed in the following examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

Example 1 Construction of a scFvFc: ζ cDNA Incorporating the CE7 V_(H)and V_(L) Sequences and Expression of the Construct in T-Cells

Based on the sequences published by Amstutz et al., PCR was carried outon cDNA generated from the CE7 hybridoma and cloned V_(H) and V_(L)segments were isolated and sequenced [54]. Referring now to FIG. 2A,there is shown a schematic of the CE7R/scFvFc: ζ chimeric receptor. Afull length scFvFc: ζ cDNA designated CE7R was constructed using methodwell known in the art by PCR splice overlap extension and consists ofthe human GM-CSF receptor alpha chain leader peptide, CE7 V_(H), Gly-Serlinker, CE7 V_(L), human IgG₁ Fc, human CD4 TM, and human cytoplasmiczeta chain. The amino acid sequence of the receptor is shown in SEQ IDNO:2.

Referring now to FIG. 2B, there is shown a plasmid comprising theCE7R/scFvFc: ζ chimeric receptor in an expression vector. Using methodswell known in the art, the cDNA construct containing the CE7R/scFvFc: ζchimeric receptor was ligated into the multiple cloning site of amodified pMG plasmid (Invitrogen, San Diego) to generate pMG-CE7R/HyTk.This expression vector co-expresses the HyTK cDNA encoding hygromycinphosphotransferase for in vitro drug selection and the herpes thymidinekinase that renders cells susceptible to the cytotoxic action ofganciclovir. Expression of the CE7R scFvFc: ζ and HyTK is linked by thedicistronic mRNA configuration having an internal ribosome entry site(IRES). The scFvFc: ζ cDNA is 5′ to HyTK in pMG. “SpAn” represents asynthetic polyadenylation site with a strong pause site to limittranscriptional interference. The synthetic polyA site is based on therabbit B-globin gene using a highly conserved AATAAA sequence and a GT/Trich flanking sequence downstream from the hexanucleotide sequence(109). The pause site is derived from the C2 complement gene (110). Thesymbol “bGh pAn” represents the bovine growth hormone (bGh)polyadenylation (pAn) signal and a transcriptional pause site (111). Itis used to minimize interference and possible recombination events. Thenucleotide sequence of this plasmid is shown in SEQ ID NO:5 and FIG.1A-H.

The CE7-specific scFvFc: ζ receptor protein is expressed in PrimaryHuman T cells. To determine whether the CE7-specific scFvFc: ζ constructcould be expressed as an intact chimeric protein, T cells weretransfected with the plasmid of Example 1 containing the CE7 Chimericreceptor. Linearized plasmid was electroporated under optimizedconditions and stable transfectants selected by addition of hygromycinto cultures. Referring now to FIG. 3, there are shown the results ofWestern blot analyses of T-cells transfected with the CE7R/scFvFc: ζchimeric receptor in an expression vector of the present invention.Using methods known in the art, whole cell lysates from mocktransfectants (cells containing the pMG plasmid without the CE7R/scFvFc:ζ chimeric receptor), T-cells transfected with the CE7R/scFvFc: ζchimeric receptor, and T-cells transfected with another chimericreceptor (Anti CD20 scFvFc: ζ) were examined. Western blot of whole celllysates with an anti-zeta antibody probe shows both the endogenous zetafragment and the expected intact 66-kDa chimeric receptor protein isexpressed in cells transfected with a chimeric receptor but not in cellstransfected with plasmid lacking the DNA constructs of the presentinvention.

Referring now to FIG. 4, there are shown the results of flow cytometricanalysis of the transfected cells of the present invention. Usingmethods known in the art and discussed in detail in the followingExamples, flow cytometric analysis of transfected T-cells reacted withanti-murine FAB which react with the CE7 portion of the CE7R andanti-human Fc specific antibodies which react with the IgG portion ofthe CE7R, confirmed the cell-surface expression of the CE7R scFvFc: ζ onT cell transfectants as evidenced by cosegregation of the CE7R antigenswith known T cell antigens.

Example 2 Anti-Neuroblastoma Effector Functions of T Cells Expressingthe CE7R Chimeric Immunoreceptor

IL-2 Production

Referring now to FIG. 5, there is a graphical representation which showsthe production of IL-2 by T-cells expressing the CE7R/scFvFc: ζ chimericreceptor that are co-cultured with neuroblastoma cells. Using techniquesknown to those skilled in the art and discussed in detail in thefollowing examples, the function of the CE7R chimeric immunoreceptor inT cells was first assessed by expressing this scFvFc: ζ construct inJurkat T cells. CE7R+ Jurkat transfectants produced IL-2 whenco-cultured with a panel of neuroblastoma cell lines. IL-2 productionwas antigen specific as evidenced by the observations that mocktransfected Jurkat cells are not activated to produce IL-2 when exposedto the same neuroblastoma stimulators and that IL-2 production wasinhibited in a dose-dependent fashion by the addition to culture ofsoluble CE7 mAb.

Cytotolytic Activity

Referring now to FIGS. 6 A-D, there are shown graphical representationsthe anti-neuroblastoma activity of CD8⁺ cells expressing theCE7R/scFvFc: ζ chimeric receptor. Primary human CD4⁺ and CD8⁺ T cellclone transfectants expressing the CE7R have been generated usingtechniques well known in the art and discussed further in detail in thefollowing Examples. Like Jurkat transfectants, CD4⁺ and CD8⁺ clonessecrete cytokines (IFN-γ and gm-CSF) specifically upon co-culture withhuman neuroblastoma cells. Moreover, CE7R⁺ CD8⁺CTL clones display highlevels of cytolytic activity in standard 4-hr chromium release assaysagainst human neuroblastoma cell lines yet do not kill primary humanfibroblasts (“Fibros” in FIG. 6) or other tumor lines that are devoid ofthe CE7 epitope (“K562” in FIG. 6).

Example 3 Generation and Characterization of T Cell Clones forTherapeutic Use

All T cells administered are TCR a/b⁺ CD4⁻CD8⁺ scFvFc: ζ⁺ T cell clonescontaining unrearranged chromosomally integrated plasmid DNA. T cellsare isolated from the peripheral blood of patient's withrecurrent/refractory neuroblastoma. Materials and methods employed toisolate, genetically modify, and expand CD8⁺ T cell clones from healthymarrow donors are detailed in Examples 4-8. T cell clones geneticallymodified to express the CE7R scFvFc: ζ chimeric immunoreceptor and HyTKare selected for:

-   -   a. TCRa/b⁺, CD4⁻, CD8⁺ surface phenotype as determined by flow        cytometry.    -   b. Presence of a single copy of chromosomally integrated plasmid        vector DNA as evidenced by Southern blot.    -   c. Expression of the scFvFc: ζ gene product as detected by        Western blot.    -   d. Specific lysis of human CE7⁺ cell lines in 4-hr chromium        release assays.    -   e. Dependence on exogenous IL-2 for in vitro growth.    -   f. Mycoplasma, fungal, bacterial sterility and endotoxin levels        <5 EU/ml.    -   g. In vitro sensitivity of clones to ganciclovir.

Example 4 Materials for Isolating, Genetically Modifying and ExpandingCD8⁺ T Cell Clones from Healthy Marrow Donors for Therapeutic Use

1. Culture Media and Media Supplements

Culture media used in the studies include RPMI 1640 HEPES (IrvineScientific, Irvine, Calif.) for all cell cultures. All media ispurchased in 0.5 liter bottles and meets current FDA guidelines for usein adoptive immunotherapy studies in humans. Supplements to the culturemedia include L-glutamine (BioWhittaker, Walkersville, Md.) and fetalcalf serum (Hyclone, Logan, Utah) heat inactivated at 56° C. for 30minutes. All reagents are shipped to CRB-3008, inspected, and stored at−20° C. or 4° C. as appropriate for the reagent.

2. OKT3

Orthoclone OKT3 (Ortho) 1 mg/ml is aliquoted into sterile cryovials arestored at −20° C. in CRB-3008 until thawed for study subject T cellexpansion.

3. Interleukin 2

Pharmaceutical grade recombinant human Interleukin-2 (rhIL-2)(Proleukin, Chiron, Emeryville, Calif.) is supplied in vials containing0.67 mg of lyophilized IL-2 and having a specific activity of 1.5×10⁶IU/mg protein. The lyophilized recombinant IL-2 is reconstituted withsterile water for infusion and diluted to a concentration of 5×10⁴units/ml. IL-2 is aliquoted into sterile vials and stored at −20° C. inCRB-3008. rhIL-2 for direct patient administration is dispensed perstandard practice.

4. Plasmid DNA

The plasmid pMG-CE7R/HyTK containing the CE7-specific scFvFc: ζ cDNA andHyTK cDNA constructs is manufactured under GLP conditions Ampulescontaining 100 mg of sterile plasmid DNA in 40 ml of pharmaceuticalwater. Vector DNA is stored in a −70° C. freezer in CRB-3008.

5. Hygromicin

The mammalian antibiotic hygromycin is used to select geneticallymodified T cells expressing the HyTK gene. Commercially availablehygromycin (Invitrogen, San Diego, Calif.) is prepared as a sterilesolution of 100 mg/ml active drug and is stored at 4° C. in CRB-3008.

6. EBV-Induced B Cell Lines

Lymphoblastoid cell lines (LCL) are necessary feeder cells for T cellexpansion and have been used for this purpose in FDA-approved clinicaladoptive therapy trials. An EBV-induced B cell line designated TM-LCLwas established from a healthy donor by co-culture of PBMC withsupernatants of the B95-8 cell line (American Type Culture Collections)in the presence of cyclosporin A. This cell line is used as anirradiated feeder cell line. This cell line has tested negative foradventitious microorganisms as well as EBV production by cord bloodtransformation assay. Working stocks of TM-LCL have been cyropreservedin CRB-3008 after transfer. These stocks have been thawed and retestedfor bacterial, fungal and mycoplasma sterility. TM-LCL feeder cells areirradiated to 8,000 cGy prior to co-culture with T cells.

7. Feeder PBMCs

Allogeneic PBMC isolated from healthy donors meeting Blood Bank criteriaand laboratory screening for clinical cell product donation areharvested by leukapheresis and supplied to CRB 3008 in a collection bagfollowing irradiation to 3,300 cGy. This apheresis product is thencyropreserved in ampules containing 50×10⁶ mononuclear cells in theCRB-3008 liquid nitrogen tank.

Example 5 Generation of CD8⁺ CTL Clones Genetically Modified to Expressthe CE7-Specific scFvFc: ζ Receptor (CE7R) and HyTK

1. Peripheral Blood Lymphocytes—Collection and Separation

Peripheral blood mononuclear cells (PBMC) are obtained from the studysubject's designated marrow donor by leukapheresis. The mononuclearcells are separated from heparinized whole blood by centrifugation overclinical grade Ficoll (Pharmacia, Uppsula, Sweden). PBMC are washedtwice in sterile phosphate buffered saline (Irvine Scientific) andsuspended in culture media consisting of RPMI, 10% heat inactivated FCS,and 4 mM L-glutamine.

2. Activation of PBMC

T cells present in patient PBMC are polyclonally activated by additionto culture of Orthoclone OKT3 (30 ng/ml). Cell cultures are thenincubated in vented T75 tissue culture flasks in the study subject'sdesignated incubator. Twenty-four hours after initiation of culturerhIL-2 is added at 25 U/ml.

3. Genetic Modification of Activated PBMC

Three days after the initiation of culture PBMC are harvested,centrifuged, and resuspended in hypotonic electroporation buffer(Eppendorf) at 20×10⁶ cells/ml. 25 mg of plasmid DNA together with 400ml of cell suspension are added to a sterile 0.2 cm electroporationcuvette. Each cuvette is subjected to a single electrical pulse of250V/40 ms delivered by the Multiporator (Eppendorf) then incubated forten minutes at room temperature. Following the RT incubation, cells areharvested from cuvettes, pooled, and resuspended in phenol red-freeculture media containing 25 U/ml rhIL-2. Flasks are placed in thepatient's designated tissue culture incubator. Three days followingelectroporation hygromycin is added to cells at a final concentration of0.2 mg/ml. Electroporated PBMC are cultured for a total of 14 days withmedia and IL-2 supplementation every 48-hours.

4. Cloning of Hygromycin-Resistant T Cells

The cloning of hygromycin-resistant CD8⁺CTL from electroporatedOKT3-activated patient PBMC is initiated on day 14 of culture. Cellsexpressing FvFc product are positively selected for using antibodies toFab and Fc and/or Protein A-FITC label using techniques well known inthe art. Following incubation of electroporated cells with Fab and Fcantibody or Protein A-FITZ, cells expressing the FvFc are isolated byimmunogenetic beads or colummns or fluorescent activated cel sortingprocedures. Viable patient PBMC are added to a mixture of 100×10⁶cyropreserved irradiated feeder PBMC and 20×10⁶ irradiated TM-LCL in avolume of 200 ml of culture media containing 30 ng/ml OKT3 and 50 U/mlrhIL-2. This mastermix is plated into ten 96-well cloning plates witheach well receiving 0.2 ml. Plates are wrapped in aluminum foil todecrease evaporative loss and placed in the patient's designated tissueculture incubator. On day 19 of culture each well receives hygromycinfor a final concentration of 0.2 mg/ml. Wells are inspected for cellularoutgrowth by visualization on an inverted microscope at Day 30 andpositive wells are marked for restimulation.

5. Expansion of Hygromycin-Resistant Clones with CE7 Re-DirectedCytotoxicity

The contents of each cloning well with cell growth and cytolyticactivity by screening chromium release assay are individuallytransferred to T25 flasks containing 50×10⁶ irradiated PBMC, 10×10⁶irradiated LCL, and 30 ng/ml OKT3 in 25 mls of tissue culture media. Ondays 1, 3, 5, 7, 9, 11, and 13 after restimulation flasks receive 50U/ml rhIL-2 and 15 mls of fresh media. On day 5 of the stimulation cycleflasks are also supplemented with hygromycin 0.2 mg/ml. Fourteen daysafter seeding cells are harvested, counted, and restimulated in T75flasks containing 150×10⁶ irradiated PBMC, 30×10⁶ irradiated TM-LCL and30 ng/ml OKT3 in 50 mls of tissue culture media. Flasks receiveadditions to culture of rhIL-2 and hygromycin as outlined above.

6. Characterization of Hygromycin-Resistant CTL Clones

a. Cell Surface Phenotype

CTL selected for expansion for use in therapy are analyzed byimmunofluorescence on a FACSCalibur housed in CRB-3006 usingFITC-conjugated monoclonal antibodies WT/31 (αβTCR), Leu 2a (CD8), andOKT4 (CD4) to confirm the requisite phenotype of clones (abTCR⁺, CD4⁻,and CD8⁺). Criteria for selection of clones for clinical use includeuniform TCR ab⁺, CD4⁻, CD8⁺ as compared to isotype controlFITC-conjugated antibody.

b. Chromosomal Integration of Plasmid

A single site of plasmid vector chromosomal integration was confirmed bySouthern blot analysis. DNA from genetically modified T cell clones wasscreened with a DNA probe specific for the plasmid vector. TheHyTK-specific DNA probe was the 420 basepair MscI/NaeI restrictionfragment isolated from pMG-CE7R/HyTK. Probe DNA was ³²p labeled using arandom primer labeling kit (Boehringer Mannheim, Indianapolis, Ind.). Tcell genomic DNA was isolated per standard technique. Ten micrograms ofgenomic DNA from T cell clones was digested overnight at 37° C. with 40units of XbaI and HindIII and then electrophoretically separated on a0.85% agarose gel. DNA was then transferred to nylon filters (BioRad,Hercules, Calif.) using an alkaline capillary transfer method. Filterswere hybridized overnight with the HyTK-specific ³²P-labeled probe in0.5 M Na₂PO₄, pH 7.2, 7% SDS, containing 10 μg/ml salmon sperm DNA(Sigma) at 65° C. Filters were then washed four times in 40 mM Na₂PO₄,pH 7.2, 1% SDS at 65° C. and then visualized using a phosphoimager(Molecular Dynamics, Sunnyvale, Calif.). Criteria for clone selection isa single unique band with the HyTK probe.

c. Expression of the CE7-Specific scFvFc: ζ Receptor

Expression of the CE7R scFvFc: ζ receptor is determined by Western blotprocedure in which chimeric receptor protein is detected with ananti-zeta antibody. Whole cell lysates of transfected T cell clones weregenerated by lysis of 2×10₇ washed cells in 1 ml of RIPA buffer (PBS, 1%NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 tablet/10 mlComplete Protease Inhibitor Cocktail (Boehringer Mannheim). After aneighty minute incubation on ice, aliquots of centrifuged whole celllysate supernatant were harvested and boiled in an equal volume ofloading buffer under reducing conditions then subjected to SDS-PAGEelectrophoresis on a precast 12% acrylamide gel (BioRad). Followingtransfer to nitrocellulose, membranes were blocked in blotto solutioncontaining 0.07 gm/ml non-fat dried milk for 2 hours. Membranes werewashed in T-TBS (0.05% Tween 20 in Tris buffered saline pH 8.0) thenincubated with primary mouse anti-human CD3ζ monoclonal antibody 8D3(Pharmingen, San Diego, Calif.) at a concentration of 1 mg/ml for 2hours. Following an additional four washes in T-TBS, membranes areincubated with a 1:500 dilution of goat anti-mouse IgG alkalinephosphatase-conjugated secondary antibody for 1 hour. Prior todeveloping, membranes were rinsed in T-TBS then developed with 30 ml of“AKP” solution (Promega, Madison, Wis.) per the manufacturer'sinstructions. Criteria for clone selection is the presence of a 66 kDachimeric zeta band.

d. Cytolytic Specificity for CE7+ Cells and Lack of Cytolytic ActivityAgainst Recipient Fibroblasts Activity

CD8⁺ cytotoxic T cell clones expressing the CE7R scFvFc: ζ receptorrecognize and lyse human CE7⁺ target cells following interaction of thechimeric receptor with the cell surface target epitope in an HLAunrestricted fashion. The requirements for target cell CE7 expressionand class IMHC independent recognition are confirmed by assaying eachαβTCR⁺, CD8⁺, CD4⁻, CE7R⁺ CTL clones against a panel of MHC-mismatchedhuman neuroblastoma cell lines (KCNR, Be-2) as well as the CE7⁻ lineK562 (a CE7-negative, NK-sensitive target) and recipient fibroblasts. Tcell effectors are assayed 12-14 days following stimulation with OKT3.Effectors are harvested, washed, and resuspended in assay media;2.5×10⁵, 1.25×10⁵, 0.25×10⁵, and 0.05×10⁵ effectors are plated intriplicate at 37° C. for 4 hours with 5×10³ target cells in V-bottommicrotiter plates (Costar, Cambridge, Mass.). After centrifugation andincubation, 100 mL aliquots of cell-free supernatant is harvested andcounted. Percent specific cytolysis is calculated as follows:

$\frac{( {{Experimental}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{release}} ) - ( {{control}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{release}} )}{( {{Maximum}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{release}} ) - ( {{control}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{release}} )} \times 100$

Control wells contain target cells incubated in assay media. Maximum⁵¹Cr release is determined by measuring the ⁵¹Cr content of target cellslysed with 2% SDS. Criteria for clone selection is >50% specific lysisof both neuroblastoma targets at an effector:target ratio of 25:1 andless than 10% specific lysis of K562 and fibroblasts at an E:T ratio of5:1.

Example 6 Microbiologic Surveillance of T Cell Cultures

Aliquots of media from the T cell cultures are plated onto bacterial andfungal growth media every 14 days. Cultures with evident contaminationwill be immediately discarded. To detect mycoplasma contamination,aliquots are assayed every 14 days using the Gen-Probe test kit (SanDiego, Calif.) and cultures with mycoplasma contamination discarded.Prior to infusion of T cell clones and following resuspension in 0.9%saline, Gram staining will be done on the cell suspension to excludeovert contamination and endotoxin levels determined by ELISA to excludecell product re-infusion if levels are above a 5 EU/kg burden ofendotoxin is present in the cell product. T cell clones will also becyropreserved in case archival specimens are needed.

Example 7 Quality Control Criteria for Release of Clones for Re-Infusion

The criteria set forth in Table 1 must be met prior to release of Tcells for re-infusion.

TABLE 1 Criteria for Release of Clones Test for: Release Criteria:Testing Method: Viability of Clinical >90% Trypan blue exclusionPreparation Cell-Surface Phenotype Uniformly TCRa/b⁺, Flow cytometricevaluation CD4⁺, CD8⁺ with isotype controls. Vector Rearrangement Singleband Southern Blot with HyTK- Specific Probe scFvFc: ζ Expression 66-kDBand Western Blot with Human Zeta-Specific Primary AntibodyAnti-Neuroblastoma >50% Specific Lysis at E:T 4 hr-Chromium ReleaseCytolytic Activity Ratio of 25:1 Against Assay KCNR and Be-2 and <25% SLagainst K562 and fibros at an E:T of 5:1. Sensitivity to Ganciclovir<10% Cell viability After Trypan blue-exclusion cell 14-days ofco-culture in enumeration. 5 μM ganciclovir. Sterility All screeningbacterial/ Bacterial/fungal by routine fungal cultures neg for >7clinical specimen culture. days. Mycoplasma neg at Mycoplasma byGene-Probe time of cyropreservation RIA. and within 48 hrs of eachEndotoxin by ELISA. infusion. Endotoxin level Gram stain by clinical<5E.I./kg in washed cell microbiology lab. preparation. Gram stainnegative on day of re- infusion.

Example 8 Quantitative PCR for T Cell Persistence In Vivo

The duration of in vivo persistence of scFvFc:^(ζ) CD8⁺ CTL clones inthe circulation is determined by quantitative PCR (Q-PCR) utilizing therecently developed TaqMan fluorogenic 5′ nuclease reaction. Q-PCRanalysis is performed by the Cellular and Molecular Correlative Core ongenomic DNA extracted from study subject PBMC obtained prior to and ondays +1 and +7 following each T cell infusion. Following the thirdinfusion PBMC are also sampled on day +14, +21, +51 (Day +100 followingstem cell rescue). Should any study subject have detectablegene-modified T cells on day +100, arrangements are made to re-evaluatethe patient monthly until the signal is undetectable. Published datafrom Riddel et al. has determined that adoptively transferred T cellsare detected in the peripheral blood of study subjects one day followinga cell dose of 5×10⁹ cells/m² at a frequency of 1-3 cells/100 PBMC, thusthe doses of cells for this study will result in a readily detectablesignal (77). DNA is extracted from PBMC using the Qiagen QiAmp kit. Theprimers used to detect the scFvFc: ζ gene are SEQ ID NO:6:5′-TCTTCCTCTACACAGCAAGCT CACCGTGG-3′), the 5′ heavy chain Fc specificprimer, and SEQ ID NO:7: 5′-GAGGGTTCTTCCT TCCTTCTCGGCTTTC-3′), the 3′HuZeta primer, which amplify a 360 basepair fragment spanning theFc-CD4-TM-zeta sequence fusion site. The TaqMan hybridization probe isSEQ ID NO:8: 5′-TTCACTCTGAA GAAGATGCCTAGCC-3′ that is 5′FAM—3′TAMRAlabeled. A standard curve is generated from genomic DNA isolated from aT cell clone with a single copy of integrated plasmid spiked intounmodified T cells at frequencies of 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶. Acontrol primer/probe set specific for the human beta-globin gene is usedto generate a standard curve for cell number and permits the calculationof the frequency of genetically modified clone in a PBMC sample. Thebeta-globin amplimers are as follows: SEQ ID NO:9: 5′-ACACAACTGTGTTCACTAGC-3′ (Pco3) and SEQ ID NO:10:5′-GTCTCCTTAAACCTGTCTTG-3′ (GII) andthe Taqman probe is SEQ ID NO:11: 5′-ACCTGACTCCTGAGG AGAAGTCT-3′ that isHEX5′----3′TAMRA labeled. All patients will have persistence data andimmune response data to the scFvFc: ζ and HyTK genes compared todetermine if limited persistence can be attributed to the development ofan immune response to gene-modified T cells.

Example 9

1. Staging Criteria and Patient Eligibility

a. Staging Criteria

Prior to Treatment

-   -   Immunohistopathologically confirmed recurrent/refractory        neuroblastoma from tissue biopsy/marrow sample OR radiographic        demonstration of tumor growth/recurrence at previous        pathologically documented site of tumor.

CT Scan of Chest Abdomen and Pelvis with and without IV contrast.

MRI of Head with and without IV contrast.

Bone Scan and MIBG Scan (if available)

24-hour urine for HVA/VMA

Serum LDH and ferritin

Within 14 Days Prior to First T Cell Infusion

CT Scan of Chest Abdomen and Pelvis with and without IV contrast.

MRI of Head with and without IV contrast.

Bone Scan and MIBG Scan (if available providing that first scan waspositive)

24-hour urine for HVA/VMA

Serum LDH and ferritin

b. Patient Eligibility

Patient Inclusion Criteria

-   -   Recurrent disseminated neuroblastoma or disseminated        neuroblastoma that is refractory to 1^(st) line therapy.

Male or female subjects greater than 12 months of age.

Availability for peripheral blood sample drawing for study tests asoutlined in Table 2.

TABLE 2 Calender of Specific Evaluations Infusion Infusion #2 #3 DayScreening Infusion Day Day Day Day Day Day Day Day Day +56/ Day Visit #1+1 +7 +14 +15 +21 +28 +29 +35 +42 70 +100 History and X X X X X X X X XX X X X Physical/Lansky Score CBC, DIFF, PLT X X X X X X X X X X X X XChem 18 X X X X X X X X X X X X X EBV, HIV X Serologies Head CT* X PCRfor X X X X X X X X X X X X plasmid Sequence in PBMC 24 Hr Urine for XX* X X HVA/VMA Radiographic/ X* X X BMA& BMX Disease Response PeripheralX Blood for Immune Response *Studies are completed within 14 days priorto Infusion #1

2. Treatment Design and Rules for Dose Escalation

Peripheral blood mononuclear cells are obtained from patients byleukapheresis. Patient-derived T cell clones are generated from theseleukapheresis products. Each participant receives a series of threeescalating cell dose T cell infusions at two week intervals beginning assoon as clones are available and after recovery from all acuteself-limited side effects of salvage chemotherapy. The salvagechemotherapy, administered by the patient's primary oncologist, isindividualized to account for that child's previous treatment history,organ dysfunction, and disease sensitivity. The most common regimens arebe cyclophosphamide/topotecan or ifosphamide/carboplatin/etoposidecombinations, both of which have been extensively studied in childrenwith recurrent solid tumors. The first cell dose is 10⁸ cells/m², thesecond 10⁹ cells/m², and the third 10¹⁰ cells/m². Recipients optionallycan receive subcutaneously delivered low-dose rhIL-2 for ten daysbeginning 24-hrs following T cell adoptive transfer. Patients areevaluated prior to and weekly after the first infusion for a period oftwo months after which time, patients are evaluated monthly for anadditional two months. Peripheral blood is drawn at specific timesduring the study to assay for the in vivo persistence of the transferredCTL clones and the induction of anti-scFvFc: ζ and HyTK immuneresponses. In those patients with detectable tumor at the time adoptivetherapy commences, anti-tumor responses are assessed by serialradiographic studies of areas of bulky disease, and/or bone marrowcytology in those subjects with marrow infiltration, and/or tumormarkers (HVA, VMA).

3. Treatment Plan

a. Schedule of Administration of CE7R⁺, CD8⁺ T Cell Clones

A series of three escalating cell dose infusions (Table 3) can beadministered to patients at two-week intervals. T cell infusionscommence at the earliest time of their availability provided thatrecipients have recovered from the acute hematologic and toxic sideeffects of salvage chemotherapy. For those who do not meet the specifiedcriteria (detailed below) for T cell re-infusion at the time clones arefirst available, T-cell clones are cryopreserved until these criteriaare met. Clones are then thawed and undergo one two-week in vitroexpansion cycle prior to re-infusion. The initial two cell doses are ofmodest numbers and consistent with cell doses used by Greenberg andRiddell [58]. Cell dose level III is higher than previously reported forT cell clones but within cell dose range of prior LAX cell studies. Lowdose s.c. IL-2 is administered in a second cohort of five patients tosupport the in vivo persistence of transferred CTL. IL-2 injectionsbegin 24-hrs following adoptive transfer of T cell clones and continuefor ten days following the second and third T cell infusion provided nograde 3-4 toxicity is observed with the administration of the first Tcell dose or is accompanied with the second T cell dose/IL-2.

TABLE 3 CE7R⁺HyTK⁺, CD8⁺ Cytotoxic T Cell Administration ScheduleProtocol Cohort 1 Cohort 2 Cell Dose Day Cell Dose Cell Dose/IL-2 I 0 1× 10⁸ cells/m² BSA 1 × 10⁸ cells/m² BSA No IL-2 II +14 1 × 10⁹ cells/m²BSA 1 × 10⁹ cells/m² BSA s.c. IL-2 5 × 10⁵ U/m² q12 hrs × 10 days III+28 1 × 10¹⁰ cells/m² BSA 1 × 10¹⁰ cells/m² BSA s.c. IL-2 5 × 10⁵ U/m²q12 hrs × 10 days

Each infusion consists of a composite of up to five T cell clones toachieve the cell dose under study.

On the day of infusion T cell clones expanded in CRB-3008 areaseptically processed per standard technique on a CS-3000 bloodseparation device for cell washing and concentrating. Processed cellsare resuspended in 100 ml of 0.9% NaCl with 2% human serum albumin in abag suitable for clinical re-infusion.

Patients can be admitted to a hospital, for example, for their T cellinfusions and are discharged no sooner than 23 hours following theirinfusion provided that no toxicities are observed. Otherwise patientsremain hospitalized until resolution of any infusion-related toxicitydeemed to pose a significant risk to the study subject as an outpatient.

T cells are infused intravenously over 30 minutes through a central lineif available, if not an age appropriate sized I.V. catheter is insertedinto a peripheral vein. The I.V tubing does not have a filter to avoidtrapping of cells. The infusion bag is gently mixed every 5 minutesduring the infusion.

The doctor or his representative is present during the infusion andimmediately available for 2 hours following the infusion. Nursingobservation and care is employed throughout the patient's hospital stay.

Subjects' oxygen saturation is measured by continuous pulse-oximetrybeginning pre-infusion and continuing for at least 2 hrs or untilreadings return to their pre-infusion baseline.

Subjects experiencing regimen-related toxicities due to their salvagechemotherapy will have their infusion schedule delayed until thesetoxicities have resolved. The specific toxicities warranting delay of Tcell infusions include: (a) Pulmonary: Requirement for supplementaloxygen to keep saturation greater than 95% or presence of radiographicabnormalities on chest x-ray that are progressive; (b) Cardiac: Newcardiac arrhythmia not controlled with medical management. Hypotensionrequiring pressor support; (c) Active Infection: Positive blood culturesfor bacteria, fungus, or virus within 48-hours of day 0; (d) Hepatic:Serum total bilirubin, or transaminases more than 5× normal limit; (e)Renal: Serum creatinine >2.0 or if patient requires dialysis; (f)Neurologic: Seizure activity within one week preceding day 0 orclinically detectable encephalopathy or new focal neurologic deficits;(g) Hematologic: Clinically evident bleeding diathesis or hemolysis.Platelet count must be greater than 20,000 and absolute neutrophil countgreater than 500. Patients may be supported with PRBC and platelettransfusions.

b. Interleukin-2 Administration

Recombinant human IL-2 (rHuIL-2, Proleukin, Chiron, Emeryville, Calif.)resuspended for s.c. injection by standard pharmacy guidelines isadministered provided that (1) no grade 3-4 oxicities are encountered inpersons not receiving IL-2 at cell dose levels I-III and (2) no grade3-4 toxicities are observed with the first and second cell doses inpersons receiving IL-2. The initial IL-2 course is 5×10⁵ U/m²/dose q 12hrs for 10 days beginning no sooner than 24-hrs following T cellre-infusion #2 and lasting no longer than 48-hrs prior to the third Tcell dose (Day +28). A second IL-2 course of 5×10⁵ U/m²/dose q 12 hrsfor 10 days is administered no sooner than 24-hrs following the third Tcell dose provided no grade 3 or higher toxicity was encountered duringthe first IL-2 course. If grade 3-4 toxicity is encountered during thefirst IL-2 course, IL-2 is not administered following the third T celldose. Patients not receiving IL-2 who have T cell persistence data(Q-PCR) demonstrating disappearance of clones following the third T cellinfusion and those subjects receiving IL-2 who have completed theprescribed T cell infusions and IL-2 who similarly have persistence datademonstrating loss of clones in the circulation may receive furthercourses of IL-2 at the discretion of the treating physician.

c. Management of Toxcities and Complications

The management of mild transient symptoms such as have been observedwith LAK, TIL, and T cell clone infusions symptoms is as follows. (1)All patients are pre-medicated with 15 mg/kg of acetaminophen p.o. (max.650 mg.) and diphenhydramine 1 mg/kg I.V. (max dose 50 mg). (2) Fever,chills and temperature elevations >101° F. are managed with additionaltylenol as clinically indicated, 10 mg/kg ibuprofen p.o. (max 400 mg)for breakthrough fevers, and 1 mg/kg demerol I.V. for chills (max 50mg). Additional methods such as cooling blankets are employed for feversresistant to these measures. All subjects that develop fever or chillshave a blood culture drawn. Ceftriaxone 50 mg/kg I.V. (max dose 2 gms)is administered to non-allergic patients who in the opinion of thephysician in attendance appear septic; alternate antibiotic choices areused as clinically indicated. (3) Headache is managed withacetaminophen. (4) Nausea and vomiting are treated with diphenhydramine1 mg/kg I.V. (max 50 mg). (5) Transient hypotension is initially managedby intravenous fluid administration, however, patients with persistenthypotension require transfer to the intensive care unit for definitivemedical treatment. (6) Hypoxemia is managed with supplemental oxygen.

Patients receive ganciclovir if grade 3 or 4 treatment-related toxicityis observed. Parentally administered ganciclovir is dosed at 10mg/kg/day divided every 12 hours. A 14-day course is prescribed but maybe extended should symptomatic resolution not be achieved in that timeinterval. All patients not hospitalized at the time of presentingsymptoms are hospitalized for the first 72 hours of ganciclovir therapyfor monitoring purposes. If symptoms do not respond to ganciclovirwithin 72 hours additional immunosuppressive agents including but notlimited to corticosteroids and cyclosporin are added at the discretionof the treating physician.

d. Concomitant Therapy

All standard supportive care measures for patients undergoing therapiesare used at the discretion of the patient's physician. Active infectionsare treated according to the standard of care.

4. Toxicities Monitored and Dosage Modifications

a. Toxicities To Be Monitored

Toxicity criteria is per the NCI Common Toxicity Criteria (CTC) version2.0 for toxicity and Adverse Event Reporting. A copy of the CTC version2.0 is downloadable from the CTEP home page(http://ctep.info.nih.gov/1). All CTC guidelines apply to toxicityassessment except serum measurements of total bilirubin, ALT and AST asmany cancer patients who have recently received chemotherapy frequentlyhave prolonged elevations in bilirubin and hepatic transaminases. Forthose patients with elevated baseline serum levels of bilirubin, ALT orAST a grade 1 toxicity will be an elevation from their pre-T cellinfusion baseline up to 2.5× that baseline level. Grade 2 hepatic willbe a >2.5-5× rise from their pre-T cell infusion baseline, a grade 3toxicity >5-20× rise, and grade 4>20× baseline. Any toxicity reported byresearch participants while receiving treatment or in follow-up forwhich there is no specific CTC designation will be graded on thefollowing scale: Grade 0—no toxicity; Grade 1—mild toxicity, usuallytransient, requiring no special treatment and generally not interferingwith usual daily activities; Grade 2—moderate toxicity that may beameliorated by simple therapeutic maneuvers, and impairs usualactivities; Grade 3—severe toxicity which requires therapeuticintervention and interrupts usual activities, hospitalization may berequired or may not be required; Grade 4—life-threatening toxicity whichrequires hospitalization.

b. Criteria for Dose Modification

If a patient develops grade 2 toxicity with dose level I, the secondcell dose for that patient remains at T cell dose level I. Only if themaximal toxicity observed with the second infusion is limited to grade 2will the third and final cell dose be advanced to 10⁹ cells/m². Forthose persons requiring dose modification at cell infusion #2 whoexperience a grade 3 or greater toxicity with the second infusion, thethird infusion is cancelled. If the first grade 2 toxicity occurs withthe second cell dose, the third cell dose is held at dose level II.

c. Criteria for Removal of Patient from Treatment

If any patient develops grade 3 or higher toxicity, IL-2 if beingadministered, is stopped. Ganciclovir treatment as outlined above isinitiated at the time a grade 3 or higher toxicity is encountered inthose patients not receiving IL-2. For those patients receiving IL-2,ganciclovir treatment commences within 48-hours of stopping IL-2 if theencountered toxicity has not decreased to ≦grade 2 in that timeinterval. Grade 3 injection site toxicity is managed by discontinuationof IL-2 without T cell ablation with ganciclovir provided that this isthe only Grade 3 or greater toxicity. Any patient requiring ganciclovirfor T cell ablation do not receive further cell doses but continue beingmonitored per protocol. At the discretion of the treating physician,corticosteroids and/or other immunosuppressive drugs are added toganciclovir should a more rapid tempo of resolution of severe toxicitiesbe indicated.

d. Participant Premature Discontinuation

The reasons for premature discontinuation (for example, voluntarywithdrawal, toxicity, death) are recorded on the case report form. Finalstudy evaluations are completed at the time of discontinuation.Potential reasons for premature discontinuation include: (a) thedevelopment of a life-threatening infection; (b) the judgment of thetreating physician that the patient is too ill to continue; (c)patient/family noncompliance with therapy and/or clinic appointments;(d) pregnancy; (e) voluntary withdrawal; (f) significant and rapidprogression of neuroblastoma requiring alternative medical, radiation orsurgical intervention; and (g) grade 3 or 4 toxicity judged to bepossibly or probably related to study therapy.

5. Study Parameters and Calender (Table 2)

To occur concurrently with the patient's evaluation for disease relapseand prior to commencing with salvage chemotherapy. The specificstudies/procedures include:

-   -   Review of pathologic specimens and/or radiographic studies to        confirm diagnosis of recurrent/refractory neuroblastoma.    -   Verify inclusion/exclusion criteria by history.    -   Administer the educational proctoring to the potential research        participant (³7-yrs of age) and the parent/legal guardian,        conduct the post-educational assessment.    -   Obtain informed consent.    -   Obtain EBV and HIV serologies.    -   Conduct staging studies as outlined above.    -   Obtain serum sample for HAMA analysis if patient has received        prior murine monoclonal antibody therapy.

(b) Isolation of Peripheral Blood Mononuclear Cells for the Initiationof T Cell Cultures

Patients satisfying inclusion criteria undergo a single leukapheresisprocedure prior to receiving cytoreductive chemotherapy. Theleukapheresis product is transferred to initiate T cell cultures.

(c) Day −14 to −1: Pre-T Cell Infusion Restaging

Conduct restaging studies as outlined above.

(d) Day 0: Evaluation Immediately Prior to T Cell Infusion

-   -   Review of medical status and review of systems    -   Physical examination, vital signs, weight, height, body surface        area    -   List of concomitant medications and transfusions    -   Lansky performance status (see Table 4)    -   Complete blood count, differential, platelet count    -   Chem 18    -   Blood for protocol-specific studies (see Table 2)

TABLE 4 Lansky Scale % Able to carry on 100 Fully active normalactivity; no special care needed 90 Minor restriction in physicallystrenuous play 80 Restricted in strenuous play, tires more easily,otherwise active Mild to moderate 70 Both greater restrictions of, andless time restriction spent in active play 60 Ambulatory up to 50% oftime, limited active play with assistance/supervision 50 Considerableassistance required for any active play; fully able to engage in quietplay Moderate to 40 Able to initiate quiet activities severe restriction30 Needs considerable assistance for quiet activity 20 Limited to verypassive activity initiated by others (e.g. TV) 10 Completely disabled,not even passive play

(e) Days 0, +14, +28: Clinical Evaluation During and after T CellInfusions

Prior to the Infusion:

-   -   Interval History and Physical Exam    -   Blood draw for laboratory studies (see Table 2)

During the infusion:

-   -   Vital signs at time 0, and every 15 minutes during the infusion,        continuous pulse oximetery

Following the T cell infusion:

-   -   Vital Signs hourly for 12 hours    -   Oxygen saturation will be monitored for 2 hours following T cell        infusions. Values will be recorded prior to initiating the        infusion, immediately post-infusion, and 2 hours post-infusion.        In addition, values will be recorded every 15 minutes if they        fall below 90% until the patient recovers to his/her pre        infusion room-air baseline saturation.    -   Events will be managed by standard medical practice.

Prior to Discharge:

-   -   Interval History and Physical Exam    -   Blood draw for laboratory studies (see Table 2)

(f) Days +1, +7, +15, +21, +29, +35, +42, +56, +70, +100

-   -   Interval History and Physical Exam    -   Blood draw for CBC, diff, plt, and Chem 18    -   3 cc/kg pt wt of heparinized (preservative-free heparin 10        U/10 ml) blood sent to CRB-3002 for direct assay of peripheral        blood lymphocytes for vector DNA by PCR

(g) Days −14-0, +42, +100

-   -   CT Scan of Sites of Recurrent Disease/MRI Head/Bone/MIBG Scans:

(h) Bone Marrow Aspirate and Biopsy: Days −14-0, +56, +100

(i) Days −1, +42, +100

-   -   24-hour urine collection for HVA, VMA

(j) If a research participant is taken off treatment after receiving Tcells, restaging bone marrow evaluation will be evaluated 28 days and 56days following the last T cell dose administered.

6. Criteria for Evaluation and Endpoint Definitions

(a) Criteria for Evaluation

The phase I data obtained at each clinical assessment is outlined inTable 2. The following toxicity and adverse event determination will bemade: (a) symptoms and toxicities are evaluated as described above; (b)physical exam and blood chemistry/hematology results; and (c) adverseevent reporting

(b) Disease Status

At each disease assessment outlined in Table 2 the determination ofmeasurable disease is recorded as follows: (1) bidimensional measures ofpalpable disease and (2) on days +42 and +100 CT scans/MRI, bone scans,bone marrow studies, and Urine VMA/HVA determinations are evaluated andresponses graded per the INSS Response Criteria (Table 5).

TABLE 5 INSS Response Criteria Definition of Response to TreatmentResponse Primary Metastases Markers Complete No tumor No tumor (chest,HVA/VMA Response abdomen, liver, normal (CR) bone, bone marrow, nodes,etc.) Very Good Reduction >90% No tumor (as above HVA/VMA Partial but<100% except bone); no decreased Response improved new bonelesions, >90% (VGPR) all pre-existing Partial Reduction No new lesions;50-90% HVA/VMA Response 50-90% reduction in decreased (PR) measurablesites; 0-1 50-90% bone marrow samples with tumor; bone lesions same asVGPR Minor No new lesions; >50% reduction of any measurable lesionResponse (primary or metastases) with <50% reduction in any other; (MR)or <25% increase in any existing lesion.# Stable No new lesions; <50%reduction but <25% increase in any disease existing lesion.# (SD)Progressive Any new lesion; increase of any measurable lesion beDisease >25%; previous negative marrow positive for tumor (PD)#Quantitative immunohistochemical assessment does not apply to marrowdisease. Shrinkage in primary tumor or metastatic sites must last 4weeks to be considered a response.

7. Reporting Adverse Events

Any sign, symptom or illness that appears to worsen during the studyperiod regardless of the relationship to the study agent is an adverseevent. All adverse events occurring during treatment, whether or notattributed to the agent, that are observed by the physician. Attributesinclude a description, onset and resolution date, duration, maximumseverity, assessment of relationship to the treatment agent or othersuspect agent(s), action taken and outcome. Toxicities are scoredaccording to a 0-4 scale based on the criteria delineated in the CommonToxicity Criteria (CTC) Version 2.0 (see above). Association orrelatedness to the treatment are graded as follows: 1=unrelated,2=unlikely, 3=possibly, 4=probably, and 5=definitely related.

Unexpected adverse events are those which: (a) are not previouslyreported with adoptive T cell therapy and (b) are symptomatically andpathophysiologically related to a known toxicity but differ because ofgreater severity or specificity.

Appropriate clinical, diagnostic, and laboratory measures to attempt todelineate the cause of the adverse reaction in question must beperformed and the results reported. All tests that reveal an abnormalityconsidered to be related to adoptive transfer will be repeated atappropriate intervals until the course is determined or a return tonormal values occurs.

8. Statistical Considerations

The type and grade of toxicities noted during therapy are summarized foreach dose level. All adverse events noted by the investigator aretabulated according to the affected body system. Descriptive statisticsare used to summarize the changes from baseline in clinical laboratoryparameters. For those patients with measurable tumor at the time T celltherapy commences, responses are stratified per the INSS responsecriteria (Table 5) and summarized. Kaplan-Meier product limitmethodology are used to estimate the survival. 95% confidence intervalsare calculated for all described statistics.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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1. A CE7-specific chimeric T cell receptor comprising an intracellularsignaling domain, a transmembrane domain and an extracellular domain,the extracellular domain comprising a CE7-specific receptor and whereinthe CE7-specific chimeric T cell receptor comprises amino acids 21-631of SEQ ID NO:2.